Publication: Expression and refolding of Omp38 from Burkholderia pseudomallei and Burkholderia thailandensis, and its function as a diffusion porin
Issued Date
2004-12-15
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ISSN
02646021
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2-s2.0-10944239453
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Mahidol University
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SCOPUS
Bibliographic Citation
Biochemical Journal. Vol.384, No.3 (2004), 609-617
Suggested Citation
Jaruwan Siritapetawee, Heino Prinz, Chartchai Krittanai, Wipa Suginta Expression and refolding of Omp38 from Burkholderia pseudomallei and Burkholderia thailandensis, and its function as a diffusion porin. Biochemical Journal. Vol.384, No.3 (2004), 609-617. doi:10.1042/BJ20041102 Retrieved from: https://repository.li.mahidol.ac.th/handle/20.500.14594/21110
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Title
Expression and refolding of Omp38 from Burkholderia pseudomallei and Burkholderia thailandensis, and its function as a diffusion porin
Abstract
In the present paper, we describe cloning and expression of two outer membrane proteins, BpsOmp38 (from Burkholderia pseudomallei) and BthOmp38 (from Burkholderia thailandensis) lacking signal peptide sequences, using the pET23d(+) expression vector and Escherichia coli host strain Origami(DE3). The 38 kDa proteins, expressed as insoluble inclusion bodies, were purified, solubilized in 8 M urea, and then subjected to refolding experiments. As seen on SDS/PAGE, the 38 kDa band completely migrated to ∼110 kDa when the purified monomeric proteins were refolded in a buffer system containing 10% (w/v) Zwittergent® 3-14, together with a subsequent heating to 95 °C for 5 min. CD spectroscopy revealed that the 110 kDa proteins contained a predominant β-sheet structure, which corresponded completely to the structure of the Omp38 proteins isolated from B. pseudomallei and B. thailandensis. Immunoblot analysis using anti-BpsOmp38 polyclonal antibodies and peptide mass analysis by MALDI-TOF (matrix-assisted laser-desorption ionization-time-of-flight) MS confirmed that the expressed proteins were BpsOmp38 and BthOmp38. The anti-BpsOmp38 antibodies considerably exhibited the inhibitory effects on the permeation of small sugars through the Omp38-reconstituted liposomes. A linear relation between relative permeability rates and Mr of neutral sugars and charged antibiotics suggested strongly that the in vitro re-assembled Omp38 functioned fully as a diffusion porin.